GbpA

ABSTRACT

The invention provides gbpA polypeptides and DNA (RNA) encoding gbpA polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing gbpA polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.08/915,107, filed Aug. 20, 1997 now U.S. Pat. No. 5,885,805.

This application claims benefit of U.S. Provisional Application No.60/027,032, filed Sep. 24, 1996.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the GTP-Binding protein encoding family, hereinafterreferred to as “gbpA”.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past 20 years. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains which are resistant to some or all of thestandard antibiotics. This has created a demand for both newanti-microbial agents and diagnostic tests for this organism.

GTP-Binding proteins are often associated with essential functions inthe bacterial cell (see: Britton R A, Powell B S, Court D L, Lupski J RJ Bacteriol [1997] Jul.; 179(14):4575-4582).

Substantial effort has been invested this century in the successfuldiscovery and development of antibacterials. Paradoxically althoughantibacterials are devised to eradicate infection in mammals we knowalmost nothing of the physiology of bacterial pathogens in infectivesituations in the host. Using sequences from the Staphylococcus aureuschromosome we have developed an RT-PCR based procedure which allows usto identify those bacterial genes transcribed at any stage of infectionand also from different niches of infection. The derivation of suchinformation is a critical first step in understanding the globalresponse of the bacterial gene complement to the host environment. Fromthe knowledge of bacterial genes both of known and unknown functionwhich are widely transcribed in the host it is possible to attempt toascertain by database searching those which are present only in theeubacteria. Further prioritisation of such genes by consideration of thelikely role of their products towards the maintenance of infection andthe facility of setting up a screen for inhibitors of the biochemicalfunction indicated by their homology to characterised genes allows thecompilation of a shortlist for gene essentiality studies using geneticdeletion or controlled regulation techniques. The proteins expressed bygenes shown to be necessary for growth in vitro or in pathogenesis inanimal models provide novel targets for antibacterial screening to findagents which are broadly inhibitory towards pathogenesis. This inventionprovides S. aureus WCUH 29 polynucleotides which are transcribed ininfected tissue, in particular in both acute and chronic infections.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown protein. See SWISS-PROT: locus YYAF_BACSU, accession P37518

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel gbpA polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2] and a known amino acidsequence or sequences of other proteins such as that reported atSWISS-PROT, accession P37518, relating to a protein of locus YYAF_BACSU.

It is a further object of the invention to provide polynucleotides thatencode gbpA polypeptides, particularly polynucleotides that encode thepolypeptide herein designated gbpA.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding gbpA polypeptides comprisingthe sequence set out in Table 1 [SEQ ID NO:1] which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel gbpA protein from Staphylococcus aureus comprising the amino acidsequence of Table 1[SEQ ID NO:2], or a variant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in the depositedstrain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding gbpA, particularly Staphylococcus aureus gbpA,including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of gbpA and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStaphylococcus aureus referred to herein as gbpA as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of gbpA polypeptide encoded by naturally occurring alleles ofthe gbpA gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned gbpA polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing gbpAexpression, treating disease, for example, disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), assaying genetic variation, and administeringa gbpA polypeptide or polynucleotide to an organism to raise animmunological response against a bacteria, especially a Staphylococcusaureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to gbpA polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against gbpA polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided gbpA agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a gbpA polynucleotide or a gbpA polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Inforinatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffm, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% “identity” to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously , by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.EnzymoL. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel gbpA polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel gbpA of Staphylococcusaureus, which is related by amino acid sequence homology to a protein oflocus YYAF_BACSU. See SWISS-PROT, accession P37518. The inventionrelates especially to gbpA having the nucleotide and amino acidsequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2]respectively, and to the gbpA nucleotide sequences of the DNA in thedeposited strain and amino acid sequences encoded thereby.

TABLE 1 (A) Sequences from Staphlococcus aureus gbpA polynucleotidesequence [SEQ ID NO:1]. 5′-1 ATGCATGAAG CACATCCGGA TGTAGATATT TATATTGCTGCAGGTATCGT 51 TGGCTTACCA AACGTTGGTA AATCAACATT ATTTAATGCA ATTACAAAGG 101CGGGTGCCTT GGCAGCAAAC TATCCGTTCG CAACTATAGA CCCAAATGTG 151 GGTATTGTAGAAGTGCCAGA TGCAAGACTA CTTAAATTAG AAGAAATGGT 201 TCAACCTAAA AAAACATTACCTACTACATT TGAATTTACA GATATTGCCG 251 GAATTGTTAA AGGTGCTTCT AAAGGTGAAGGTTTAGGTAA TAAATTCTTA 301 TCACATATTA GAGAAGTAGA TGCAATTTGT CAGGTTGTTCGTGCTTTTGA 351 CGATGATAAT GTAACACATG TAGCTGGTCG AGTTGATCCA ATTGATGACA401 TCGAAGTTAT CAATATGGAA TTAGTACTTG CAGACTTAGA ATCAGTTGAA 451AAACGCTTAC CTAGAATTGA AAAATTGGCA CGTCAAAAAG ATAAGACTGC 501 TGAAATGGAAGTGCGTATTT TAACAACTAT TAAAGAAGCT TTAGAAAATG 551 GTAAACCCGC TCGTAGTATTGACTTTAATG AAGAAGACCA AAAATGGGTG 601 AATCAAGCGC AATTACTGAC TTCTAAAAAAATGCTTTATA TCGCTAATGT 651 TGGTGAAGAT GAAATTGGTG ATGATGATAA TGATAAAGTAAAAGCGATTC 701 GTGAATATGC AGCGCAAGAA GACTCTGAAG TGATTGTTAT TAGTGCAAAA751 ATTGAAGAAG AAATTGCTAC ATTAGATGAT GAAGATAAAG AAATGTTCTT 801AGAAGATTTA GGTATCGAAG AACCAGGATT AGATCGATTA ATTAGAACAA 851 CTTATGAATTATTAGGATTA TCAAGATATT TTACTGCTGG TGTGCAAGAA 901 GTACGTGCTT GGACATTTAAACAAGGTATG ACTGCACCTC AATGTGCTGG 951 TATCATTCAT ACTGATTTTG AACGTGGATTTATCCGTGCC GAAGTAACAA 1001 GTTATGATGA CTATGTACAA TATGGCGGCG AAAGTGGCGCTAAAGAAGCG 1051 GGCAGACAAC GATTAGAAGG TAAAGAATAT ATTATGCAAG ATGGCGATAT1101 CGTTCATTTC AGATTTAATG TATAA-3′ (B) GbpA polypeptide sequencededuced from the polynucleotide sequence in this table [SEQ ID NO:2].NH₂-1 MHEAHPDVDI YIAAGIVGLP NVGKSTLFNA ITKAGALAAN YPFATIDPNV 51GIVEVPDARL LKLEEMVQPK KTLPTTFEFT DIAGIVKGAS KGEGLGNKFL 101 SHIREVDAICQVVRAFDDDN VTHVAGRVDP IDDIEVINME LVLADLESVE 151 KRLPRIEKLA RQKDKTAEMEVRILTTIKEA LENGKPARSI DFNEEDQKWV 201 NQAQLLTSKK MLYIANVGED EIGDDDNDKVKAIREYAAQE DSEVIVISAK 251 IEEEIATLDD EDKEMGLEDL GIEEPGLDRL IRTTYELLGLSTYFTAGVQE 301 VRAWTFKQGM TAPGCAGIIH TDFERGFIRA EVTSYDDYVQ YGGESGAKEA351 GRQRLEGKEY IMQDGDIVHF RFNV-COOH (C) Polynucleotide sequenceembodiments [SEQ ID NO:1]. X-(R₁)_(n)-1 ATGCATGAAG CACATCCGGA TGTAGATATTTATATTGCTG CAGGTATCGT 51 TGGCTTACCA AACGTTGGTA AATCAACATT ATTTAATGCAATTACAAAGG 101 CGGGTGCCTT GGCAGCAAAC TATCCGTTCG CAACTATAGA CCCAAATGTG151 GGTATTGTAG AAGTGCCAGA TGCAAGACTA CTTAAATTAG AAGAAATGGT 201TCAACCTAAA AAAACATTAC CTACTACATT TGAATTTACA GATATTGCCG 251 GAATTGTTAAAGGTGCTTCT AAAGGTGAAG GTTTAGGTAA TAAATTCTTA 301 TCACATATTA GAGAAGTAGATGCAATTTGT CAGGTTGTTC GTGCTTTTGA 351 CGATGATAAT GTAACACATG TAGCTGGTCGAGTTGATCCA ATTGATGACA 401 TCGAAGTTAT CAATATGGAA TTAGTACTTG CAGACTTAGAATCAGTTGAA 451 AAACGCTTAC CTAGAATTGA AAAATTGGCA CGTCAAAAAG ATAAGACTGC501 TGAAATGGAA GTGCGTATTT TAACAACTAT TAAAGAAGCT TTAGAAAATG 551GTAAACCCGC TCGTAGTATT GACTTTAATG AAGAAGACCA AAAATGGGTG 601 AATCAAGCGCAATTACTGAC TTCTAAAAAA ATGCTTTATA TCGCTAATGT 651 TGGTGAAGAT GAAATTGGTGATGATGATAA TGATAAAGTA AAAGCGATTC 701 GTGAATATGC AGCGCAAGAA GACTCTGAAGTGATTGTTAT TAGTGCAAAA 751 ATTGAAGAAG AAATTGCTAC ATTAGATGAT GAAGATAAAGAAATGTTCTT 801 AGAAGATTTA GGTATCGAAG AACCAGGATT AGATCGATTA ATTAGAACAA851 CTTATGAATT ATTAGGATTA TCAACATATT TTACTGCTGG TGTGCAAGAA 901GTACGTGCTT GGACATTTAA ACAAGGTATG ACTGCACCTC AATGTGCTGG 951 TATCATTCATACTGATTTTG AACGTGGATT TATCCGTGCC GAAGTAACAA 1001 GTTATGATGA CTATGTACAATATGGCGGCG AAAGTGGCGC TAAAGAAGCG 1051 GGCAGACAAC GATTAGAAGG TAAAGAATATATTATGCAAG ATGGCGATAT 1101 CGTTCATTTC AGATTTAATG TATAA-(R₂)_(n)-Y (D)Polypeptide sequence embodiments [SEQ ID NO:2]. X-(R₁)_(n)-1 MHEAHPDVDIYIAAGIVGLP NVGKSTLFNA ITKAGALAAN YPFATIDPNV 51 GIVEVPDARL LKLEEMVQPKKTLPTTFEFT DIAGIVKGAS KGEGLGNKFL 101 SHIREVDAIC QVVRAFDDDN VTHVAGRVDPIDDIEVINME LVLADLESVE 151 KRLPRIEKLA RQKDKTAEME VRILTTIKEA LENGKPARSIDFNEEDQKWV 201 NQAQLLTSKK MLYIANVGED EIGDDDNDKV KAIREYAAQE DSEVIVISAK251 IEEEIATLDD EDKEMFLEDL GIEEPGLDRL IRTTYELLGL STYFTAGVQE 301VRAWTFKQGM TAPQCAGIIH TDFERGFIRA EVTSYDDYVQ YGGESGAKEA 351 GRQRLEGKEYIMQDGDIVHF RFNV-(R₂)_(n)-Y (E) Sequences from Staphylococcus aureus gbpApolyynucleotide ORF sequence [SEQ ID NO:3]. 5′-1 ATGCATGAAG CACATCCGGATGTAGATATT TATATTGCTG CAGGTATCGT 51 TGGCTTACCA AACGTTGGTA AATCAACATTATTTAATGCA ATTACAAAGG 101 CGGGTGCCTT GGCAGCAAAC TATCCGTTCG CAACTATAGACCCAAATGTG 151 GGTATTGTAG AAGTGCCAGA TGCAAGACTA CTTAAATTAG AAGAAATGGT201 TCAACCTAAA AAAACATTAC CTACTACATT TGAATTTACA GATATTGCCG 251GAATTGTTAA AGGTGCTTCT AAAGGTGAAG GTTTAGGTAA TAAATTCTTA 301 TCACATATTAGAGAAGTAGA TGCAATTTGT CAGGTTGTTC GTGCTTTTGA 351 CGATGATAAT GTAACACATGTAGCTGGTCG AGTTGATCCA ATTGATGACA 401 TCGAAGTTAT CAATATGGAA TTAGTACTTGCAGACTTAGA ATCAGTTGAA 451 AAACGCTTAC CTAGAATTGA AAAATTGGCA CGTCAAAAAGATAAGACTGC 501 TGAAATGGAA GTGCGTATTT TAACAACTAT TAAAGAAGCT TTAGAAAATG551 GTAAACCCGC TCGTAGTATT GACTTTAATG AAGAAGACCA AAAATGGGTG 601AATCAAGCGC AATTACTGAC TTCTAAAAAA ATGCTTTATA TCGCTAATGT 651 TGGTGAAGATGAAATTGGTG ATGATGATAA TGATAAAGTA AAAGCGATTC 701 GTGAATATGC AGCGCAAGAAGACTCTGAAG TGATTGTTAT TAGTGCAAAA 751 ATTGAAGAAG AAATTGCTAC ATTAGATGATGAAGATAAAG AAATGTGCTT 801 AGAAGATTTA GGTATCGAAG AACCAGGATT AGATCGATTAATTAGAACAA 851 CTTATGAATT ATTAGGATTA TCAACATATT TTACTGCTGG TGTGCAAGAA901 TATCATTCAT ACTGATTTTG AACGTGGATT TATCCGTGCC GAAGTAACAA 1001GTTATGATGA CTATGTACAA TATGGCGGCG AAAGTGGCGC TAAAGAAGCG 1051 GGCAGACAACGATTAGAAGG TAAAGAATAT ATTATGCAAG ATGGCGATAT 1101 CGTTCATTTC AGATTTAATGTA-3′ (F) gbpA polypeptide sequence deduced from the polynucleotide ORFsequence in this table [SEQ ID NO:4]. NH₂-1 MHEAHPDVDI YIAAGIVGLPNVGKSTLFNA ITKAGALAAN YPFATIDPNV 51 GIVEDPDARL LKLEEMVQPK KTLPTTFEFTDIAGIVKGAS KGEGLGNKFL 101 SHIREVDAIC QVVRAFDDDN VTHVAGRVDP IDDIEVINMELVLADLESVE 151 KRLPRIEKLA RQKDKTAEME VRILTTIKEA LENGKPARSI DFNEEDQKWV201 NQAQLLTSKK MYLIANVGED EIGDDDNDKV KAIREYAAQE DSEVIVISAK 251IEEEIATLDD EDKEMGLEDL GIEEPGLDRL IRTTYELLGL STYFTAGVQE 301 VRAWTFKQGMTAPQCAGIIH TDFERGFIRA EVTSYDDYVQ YGGESGAKEA 351 GRQRLWGKEY IMQDGDIVHFRFNV-COOH

Deposited Materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, and isreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain.” The depositedstrain contains the full length gbpA gene. The sequence of thepolynucleotides contained in the deposited strain, as well as the aminoacid sequence of the polypeptide encoded thereby, are controlling in theevent of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1[SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of gbpA, and also those which have at least 73% identity to apolypeptide of Table 1 [SEQ ID NOS:2 and 4] or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNOS:2 and 4], and more preferably at least 90% similarity (morepreferably at least 90% identity) to a polypeptide of Table 1 [SEQ IDNOS:2 and 4] and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to a polypeptide of Table 1 [SEQID NOS:2 and 4] and also include portions of such polypeptides with suchportion of the polypeptide generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) [SEQ ID NO:2] wherein, at the amino terminus, X is hydrogen,and at the carboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is anyamino acid residue, and n is an integer between 1 and 1000. Any stretchof amino acid residues denoted by either R group, where R is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with gbpA polypeptides fragments maybe “free-standing,” or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments-include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NOS:2 and 4], orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of gbpA, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a ftnction essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the gbpA polypeptide havinga deduced amino acid sequence of Table 1 [SEQ ID NOS:2 and.4] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NOS: 1 and 3], a polynucleotide of theinvention encoding gbpA polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 [SEQ ID NOS: 1 and 3],typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO:1] was discovered in a DNA library derivedfrom Staphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 [SEQ ID NOS:1] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NO:2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The polynucleotide of SEQ ID NO: 1,between nucleotide number 1 through number 1122 encodes the polypeptideof SEQ ID NO:2. The stop codon begins at nucleotide number 1123 of SEQID NO:1.

GbpA of the invention is structurally related to other proteins of theGTP-Binding protein encoding family, as shown by the results ofsequencing the DNA encoding gbpA of the deposited strain. The proteinexhibits greatest homology to a protein encoded at locus YYAF_BACSU,among known proteins. See SWISS-PROT accession P37518. GbpA of Table 1[SEQ ID NO:2] has about 72% identity over its entire length and about84% similarity over its entire length with the amino acid sequence ofthe polypeptide of locus YYAF_BACSU (SWISS-PROT, Accession P37518).

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence.

The polynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.NatL Acad. Sci., USA 86. 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to to 1122 or 1125 set forth in SEQ ID NO:1 ofTable 1 which encode the gbpA polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C)[SEQ ID NO:1] wherein, at the 5′ end of the molecule, X ishydrogen, and at the 3′ end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus gbpA havingthe amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodinggbpA variants, that have the amino acid sequence of gbpA polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, that do not alter the properties and activitiesof gbpA.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding gbpA polypeptide having an amino acid sequence set out in Table1 [SEQ ID NOS:2 and 4], and polynucleotides that are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding gbpA polypeptide ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 [SEQ ID NO:1].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 or SEQ ID NO:3 under stringent hybridization conditions witha probe having the sequence of said polynucleotide sequence set forth inSEQ ID NO:1 or a fragment thereof; and isolating said DNA sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding gbpA and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the gbpA gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the gbpA gene may be isolated byscreening using the DNA sequence provided in SEQ ID NO: 1 to synthesizean oligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the -polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; flugal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the gbpA polynucleotides ofthe invention for use as diagnostic reagents. Detection of gbpA in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also“individual(s)”), particularly mammals, and especially humans, infectedwith an organism comprising the gbpA gene may be detected at the nucleicacid level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled gbpA polynucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by Nasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g., Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and SI protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401(1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding gbpA can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 2.

TABLE 2 Primers for amplification of gbpA polynucleotides SEQ ID NOPRIMER SEQUENCE 5 5′-ATGTAGATAT TTATATTGCT G-3′ 6 5′-CTAATAACAATCACTTCAGA G-3′

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying gbpA DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreasedexpression of gbpA polynucleotide can be measured using any on of themethods well known in the art for the quantation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of gbpA protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a gbpAprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal, Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-gbpA or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against gbpA-polypeptide may be employedto treat infections, particularly bacterial infections and especiallydisease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theirnmediate physical interaction between pathogen and mammalian host. Thetermr “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and Agonists—Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al, CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of gbpApolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagoists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising gbpA polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a gbpA agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the gbpA polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of gbpA polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in gbpA polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for gbpA antagonists is a competitive assaythat combines gbpA and a potential antagonist with gbpA-bindingmolecules, recombinant gbpA binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. GbpA can be labeled, such as byradioactivity or a colorimetric compound, such that the number of gbpAmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing gbpA-induced activities, thereby preventing the action of gbpAby excluding gbpA from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of gbpA.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective YmRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock gbpA protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial gbpA proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with gbpA, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of gbpA, or afragment or a variant thereof, for expressing gbpA, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise. Such nucleic acid vector may comprise DNA, RNA,a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a gbpA or protein coded therefrom,wherein the composition comprises a recombinant gbpA or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid gbpA or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A gbpA polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifiing protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from. the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical. or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain gbpAprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. The sequencing data from two or more clonescontaining overlapping Staphylococcus aureus DNAs was used to constructthe contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared byroutine methods using, for example, Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of thefollowing two methods:

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch. fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2

GbpA Characterization

The determination of expression during infection of a gene fromStaphylococcus aureus entails the following procedures:

Necrotic fatty tissue from a 72 hour groin infection or an excisedkidney from an 8 day chronic kidney infection of Staphylococcus aureusWCUH29 in the mouse is efficiently disrupted and processed in thepresence of chaotropic agents and RNAase inhibitor to provide a mixtureof animal and bacterial RNA. The optimal conditions for disruption andprocessing to give stable preparations and high yields of bacterial RNAare followed by the use of hybridisation to a radiolabelledoligonucleotide specific to Staphylococcus aureus 16S RNA on Northernblots. The RNAase free, DNAase free, DNA and protein free preparationsof RNA obtained are suitable for Reverse Transcription PCR (RT-PCR)using unique primer pairs designed from the sequence of each gene ofStaphylococcus aureus WCUH29.

a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amouse animal model of infection (groin):

10 ml. volumes of sterile nutrient broth (No.2 Oxoid) are seeded withisolated, individual colonies of Staphylococcus aureus WCUH29 from anagar culture plate. The cultures are incubated aerobically (staticculture) at 37° C. for 16-20 hours. 4 week old mice (female, 18 g-22 g,strain MF1) are each infected by subcutaneous injection of 0.5 ml ofthis broth culture of Staphylococcus aureus WCUH29 (diluted in broth toapproximately 10⁸ cfu/ml.) into the anterior, right lower quadrant(groin area). Mice are monitored regularly during the first 24 hoursafter infection, then daily until termination of study. Animals withsigns of systemic infection, i.e. lethargy, ruffled appearance,isolation from group, are monitored closely and if signs progress tomoribundancy, the animal are culled immediately.

Visible external signs of lesion development are seen 24-48 hours afterinfection. Examination of the abdomen of the animal shows the raisedoutline of the abscess beneath the skin. The localised lesion remains inthe right lower quadrant, but may occasionally spread to the left lowerquadrant, and superiorly to the thorax. On occasions,.the abscess mayrupture through the overlying skin layers. In such cases, the affectedanimal is culled immediately and the tissues sampled, if possible.Failure to cull the animal may result in the necrotic skin tissueoverlying the abscess being sloughed off, exposing the abdominal musclewall.

Approximately 96 hours after infection, animals are killed using carbondioxide asphyxiation. To minimise delay between death and tissueprocessing /storage, mice should be killed individually rather than ingroups. The dead animal is placed onto its back and the fur swabbedliberally with 70% alcohol. An initial incision using scissors is madethrough the skin of the abdominal left lower quadrant, travellingsuperiorly up to, then across the thorax. The incision is completed bycutting inferiorly to the abdominal lower right quadrant. Care is takennot to penetrate the abdominal wall. Holding the skin flap with forceps,the skin is gently pulled way from the abdomen. The exposed abscess,which covers the peritoneal wall but generally does not penetrate themuscle sheet completely, is excised, taking care not to puncture theviscera

The abscess/muscle sheet and other infected tissue may require cuttingin sections, prior to flash-freezing in liquid nitrogen, therebyallowing easier storage in plastic collecting vials.

b) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amurine model of hematogenous pyelonephritis:

Overnight cultures of S. aureus WCUH29 are started from single coloniesin 5 ml of tryptic soy broth (TSB) and grown at 37° C. with shaking. Thecultures are then washed twice in sterile phosphate-buffered saline(PBS) and diluted to an A600=0.3. Male CD-1 mice (18-20 g) were infectedwith 0.2 ml of this suspension by tail vein inoculation using a 30 gaugeneedle attached to a tuberculin syringe. Each mouse receivesapproximately 4×10⁷ bacteria in this fashion. Mice are monitored dailyfor signs of illness, and usually within 48 hours show signs oflethargy, ruffled fur, sluggishness; animals which appear moribund areeuthanized prior to the end of the experiment.

All animals are euthanized via carbon dioxide overdose seven dayspost-infection. The animal is placed on its back and swabbed withethanol, and then with RNAZap, and instruments are swabbed as well. Theabdominal cavity is opened and the kidneys aseptically removed, cut intofour pieces, and placed in cryovials which are immediately frozen inliquid nitrogen.

c) Isolation of Staphylococcus aureus WCUH29 RNA from infected tissuesamples:

4-6 infected tissue samples (each approx 0.5-0.7g) in 2 ml screw-captubes are removed from −80° C. storage into a dry ice ethanol bath. In amicrobiological safety cabinet the samples are disrupted individuallywhilst the remaining samples are kept cold in the dry ice ethanol bath.To disrupt the bacteria within the tissue sample Iml of TRIzol Reagent(Gibco BRL, Life Technologies) is added followed by enough 0.1 mmzirconia/silica beads to almost fill the tube, the lid is replacedtaking care not to get any beads into the screw thread so as to ensure agood seal and eliminate aerosol generation. The sample is thenhomogenised in a Mini-BeadBeater Type BX-4 (Biospec Products). Necroticfatty tissue is strain treated for 100 seconds at 5000 rpm in order toachieve bacterial lysis. In vivo grown bacteria require longer treatmentthan in vitro grown Staphylococcus aureus bacteria which are disruptedby a 30 second bead-beat.

After bead-beating, the tubes are chilled on ice before opening in afume-hood as heat generated during disruption may degrade the TRIzol andrelease cyanide.

200 μl of chloroform is then added and the tubes shaken by hand for 15seconds to ensure complete mixing. After 2-3 minutes at room temperaturethe tubes are spun down at 12,000×g, 4° C. for 15 minutes and RNAextraction is then continued according to the method given by themanufacturers of TRIzol Reagent, i.e. the aqueous phase, approx 0.6 ml,is transferred to a sterile Eppendorf tube and 0.5 ml of isopropanol isadded. After 10 minutes at room temperature, the samples are spun at12,000 ×g, 4° C. for 10 minutes. The supernatant is removed anddiscarded, then the RNA pellet is washed with 1 ml 75% ethanol. A briefvortex is used to mix the sample before centrifuging at 7,500 ×g, 4 °C.for 5 minutes. The ethanol is removed and the RNA pellet dried undervacuum for no more than 5 minutes. Samples are then resuspended byrepeated pipetting in 100 μl of DEPC treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

RNA preparations are stored at −80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at −20 °C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1×TBE gels stained with ethidium bromide are used to visualisetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue, 1×MOPS, 2.2 M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a ³²Plabelled oligonucletide probe specific to 16S rRNA of Staphylococcusaureus (K.Greisen, M. Loeffelholz, A. Purohit and D. Leong. J. Clin.(1994) Microbiol. 32 335-351). An oligonucleotide of the sequence:5′-gctcctaaaaggttactccaccggc -3′[SEQ ID NO:7] is used as a probe. Thesize of the hybridising band is compared to that of control RNA isolatedfrom in vitro grown Staphylococcus aureus WCUH29 in the Northern blot.Correct sized bacterial 16S rRNA bands can be detected in total RNAsamples which show extensive degradation of the mammalian RNA whenvisualised on TBE gels.

d) The removal of DNA from Staphylococcus aureus WCUH29-derived RNA:

DNA was removed from 73 μl samples of RNA by a 15 minute treatment onice with 3 units of DNAaseI, amplification grade (Gibco BRL, LifeTechnologies) in the buffer supplied with the addition of 200 units ofRnasin (Promega) in a final volume of 90 μl.

The DNAase was inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 73 μl of DEPC treatedwater with the addition of Rnasin, as described in Method 1.

e) The preparation of cDNA from RNA samples derived from infectedtissue:

10 μl samples of DNAase treated RNA are reverse transcribed using aSuperScript Preamplification System for First Strand cDNA Synthesis kit(Gibco BRL, Life Technologies) according to the manufacturersinstructions. A 1 nanogram aliquot of random hexamers is used to primeeach reaction. Controls without the addition of SuperScriptIl reversetranscriptase are also run. Both +/−RT samples are treated with RNaseHbefore proceeding to the PCR reaction.

f) The use of PCR to determine the presence of a bacterial cDNA species:

PCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents: 45 μl PCR SUPERMIX (Gibco BRL, Life Technologies); 1 μl 50mM MgCl₂, to adjust final concentration to 2.5 mM; 1 μl PCR primers(optimally 18-25 basepairs in length and designed to possess similarannealing temperatures), each primer at 10 mM initial concentration; and2 μl cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows: 5 minutes at 95° C., then 50 cycles of 30 seconds each at 94°C., 42° C. and 72° C. followed by 3 minutes at 72° C. and then a holdtemperature of 4° C. The number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product, and optimally 8-30cycles if an estimation of the starting quantity of CDNA from the RTreaction is to be made; 10 μl aliquots are then run out on 1% 1×TBE gelsstained with ethidium bromide with PCR product, if present, sizesestimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5′end with a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencerusing GeneScan software as supplied by Perkin Elmer).

RT/PCR controls may include +/− reverse transcriptase reactions, 16SrRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Staphylococcus aureus WCUH29 genomicsequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStaphylococcus aureus WCUH29 total DNA. PCR reactions are set up and runas described above using approx. 1 μg of DNA in place of the CDNA and 35cycles of PCR.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: (1) Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and (2) Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in −RT controls

7 1125 base pairs nucleic acid double linear not provided 1 ATGCATGAAGCACATCCGGA TGTAGATATT TATATTGCTG CAGGTATCGT TGGCTTACCA 60 AACGTTGGTAAATCAACATT ATTTAATGCA ATTACAAAGG CGGGTGCCTT GGCAGCAAAC 120 TATCCGTTCGCAACTATAGA CCCAAATGTG GGTATTGTAG AAGTGCCAGA TGCAAGACTA 180 CTTAAATTAGAAGAAATGGT TCAACCTAAA AAAACATTAC CTACTACATT TGAATTTACA 240 GATATTGCCGGAATTGTTAA AGGTGCTTCT AAAGGTGAAG GTTTAGGTAA TAAATTCTTA 300 TCACATATTAGAGAAGTAGA TGCAATTTGT CAGGTTGTTC GTGCTTTTGA CGATGATAAT 360 GTAACACATGTAGCTGGTCG AGTTGATCCA ATTGATGACA TCGAAGTTAT CAATATGGAA 420 TTAGTACTTGCAGACTTAGA ATCAGTTGAA AAACGCTTAC CTAGAATTGA AAAATTGGCA 480 CGTCAAAAAGATAAGACTGC TGAAATGGAA GTGCGTATTT TAACAACTAT TAAAGAAGCT 540 TTAGAAAATGGTAAACCCGC TCGTAGTATT GACTTTAATG AAGAAGACCA AAAATGGGTG 600 AATCAAGCGCAATTACTGAC TTCTAAAAAA ATGCTTTATA TCGCTAATGT TGGTGAAGAT 660 GAAATTGGTGATGATGATAA TGATAAAGTA AAAGCGATTC GTGAATATGC AGCGCAAGAA 720 GACTCTGAAGTGATTGTTAT TAGTGCAAAA ATTGAAGAAG AAATTGCTAC ATTAGATGAT 780 GAAGATAAAGAAATGTTCTT AGAAGATTTA GGTATCGAAG AACCAGGATT AGATCGATTA 840 ATTAGAACAACTTATGAATT ATTAGGATTA TCAACATATT TTACTGCTGG TGTGCAAGAA 900 GTACGTGCTTGGACATTTAA ACAAGGTATG ACTGCACCTC AATGTGCTGG TATCATTCAT 960 ACTGATTTTGAACGTGGATT TATCCGTGCC GAAGTAACAA GTTATGATGA CTATGTACAA 1020 TATGGCGGCGAAAGTGGCGC TAAAGAAGCG GGCAGACAAC GATTAGAAGG TAAAGAATAT 1080 ATTATGCAAGATGGCGATAT CGTTCATTTC AGATTTAATG TATAA 1125 374 amino acids amino acidsingle linear not provided 2 Met His Glu Ala His Pro Asp Val Asp Ile TyrIle Ala Ala Gly Ile 1 5 10 15 Val Gly Leu Pro Asn Val Gly Lys Ser ThrLeu Phe Asn Ala Ile Thr 20 25 30 Lys Ala Gly Ala Leu Ala Ala Asn Tyr ProPhe Ala Thr Ile Asp Pro 35 40 45 Asn Val Gly Ile Val Glu Val Pro Asp AlaArg Leu Leu Lys Leu Glu 50 55 60 Glu Met Val Gln Pro Lys Lys Thr Leu ProThr Thr Phe Glu Phe Thr 65 70 75 80 Asp Ile Ala Gly Ile Val Lys Gly AlaSer Lys Gly Glu Gly Leu Gly 85 90 95 Asn Lys Phe Leu Ser His Ile Arg GluVal Asp Ala Ile Cys Gln Val 100 105 110 Val Arg Ala Phe Asp Asp Asp AsnVal Thr His Val Ala Gly Arg Val 115 120 125 Asp Pro Ile Asp Asp Ile GluVal Ile Asn Met Glu Leu Val Leu Ala 130 135 140 Asp Leu Glu Ser Val GluLys Arg Leu Pro Arg Ile Glu Lys Leu Ala 145 150 155 160 Arg Gln Lys AspLys Thr Ala Glu Met Glu Val Arg Ile Leu Thr Thr 165 170 175 Ile Lys GluAla Leu Glu Asn Gly Lys Pro Ala Arg Ser Ile Asp Phe 180 185 190 Asn GluGlu Asp Gln Lys Trp Val Asn Gln Ala Gln Leu Leu Thr Ser 195 200 205 LysLys Met Leu Tyr Ile Ala Asn Val Gly Glu Asp Glu Ile Gly Asp 210 215 220Asp Asp Asn Asp Lys Val Lys Ala Ile Arg Glu Tyr Ala Ala Gln Glu 225 230235 240 Asp Ser Glu Val Ile Val Ile Ser Ala Lys Ile Glu Glu Glu Ile Ala245 250 255 Thr Leu Asp Asp Glu Asp Lys Glu Met Phe Leu Glu Asp Leu GlyIle 260 265 270 Glu Glu Pro Gly Leu Asp Arg Leu Ile Arg Thr Thr Tyr GluLeu Leu 275 280 285 Gly Leu Ser Thr Tyr Phe Thr Ala Gly Val Gln Glu ValArg Ala Trp 290 295 300 Thr Phe Lys Gln Gly Met Thr Ala Pro Gln Cys AlaGly Ile Ile His 305 310 315 320 Thr Asp Phe Glu Arg Gly Phe Ile Arg AlaGlu Val Thr Ser Tyr Asp 325 330 335 Asp Tyr Val Gln Tyr Gly Gly Glu SerGly Ala Lys Glu Ala Gly Arg 340 345 350 Gln Arg Leu Glu Gly Lys Glu TyrIle Met Gln Asp Gly Asp Ile Val 355 360 365 His Phe Arg Phe Asn Val 3701122 base pairs nucleic acid double linear not provided 3 ATGCATGAAGCACATCCGGA TGTAGATATT TATATTGCTG CAGGTATCGT TGGCTTACCA 60 AACGTTGGTAAATCAACATT ATTTAATGCA ATTACAAAGG CGGGTGCCTT GGCAGCAAAC 120 TATCCGTTCGCAACTATAGA CCCAAATGTG GGTATTGTAG AAGTGCCAGA TGCAAGACTA 180 CTTAAATTAGAAGAAATGGT TCAACCTAAA AAAACATTAC CTACTACATT TGAATTTACA 240 GATATTGCCGGAATTGTTAA AGGTGCTTCT AAAGGTGAAG GTTTAGGTAA TAAATTCTTA 300 TCACATATTAGAGAAGTAGA TGCAATTTGT CAGGTTGTTC GTGCTTTTGA CGATGATAAT 360 GTAACACATGTAGCTGGTCG AGTTGATCCA ATTGATGACA TCGAAGTTAT CAATATGGAA 420 TTAGTACTTGCAGACTTAGA ATCAGTTGAA AAACGCTTAC CTAGAATTGA AAAATTGGCA 480 CGTCAAAAAGATAAGACTGC TGAAATGGAA GTGCGTATTT TAACAACTAT TAAAGAAGCT 540 TTAGAAAATGGTAAACCCGC TCGTAGTATT GACTTTAATG AAGAAGACCA AAAATGGGTG 600 AATCAAGCGCAATTACTGAC TTCTAAAAAA ATGCTTTATA TCGCTAATGT TGGTGAAGAT 660 GAAATTGGTGATGATGATAA TGATAAAGTA AAAGCGATTC GTGAATATGC AGCGCAAGAA 720 GACTCTGAAGTGATTGTTAT TAGTGCAAAA ATTGAAGAAG AAATTGCTAC ATTAGATGAT 780 GAAGATAAAGAAATGTTCTT AGAAGATTTA GGTATCGAAG AACCAGGATT AGATCGATTA 840 ATTAGAACAACTTATGAATT ATTAGGATTA TCAACATATT TTACTGCTGG TGTGCAAGAA 900 GTACGTGCTTGGACATTTAA ACAAGGTATG ACTGCACCTC AATGTGCTGG TATCATTCAT 960 ACTGATTTTGAACGTGGATT TATCCGTGCC GAAGTAACAA GTTATGATGA CTATGTACAA 1020 TATGGCGGCGAAAGTGGCGC TAAAGAAGCG GGCAGACAAC GATTAGAAGG TAAAGAATAT 1080 ATTATGCAAGATGGCGATAT CGTTCATTTC AGATTTAATG TA 1122 374 amino acids amino acidsingle linear not provided 4 Met His Glu Ala His Pro Asp Val Asp Ile TyrIle Ala Ala Gly Ile 1 5 10 15 Val Gly Leu Pro Asn Val Gly Lys Ser ThrLeu Phe Asn Ala Ile Thr 20 25 30 Lys Ala Gly Ala Leu Ala Ala Asn Tyr ProPhe Ala Thr Ile Asp Pro 35 40 45 Asn Val Gly Ile Val Glu Val Pro Asp AlaArg Leu Leu Lys Leu Glu 50 55 60 Glu Met Val Gln Pro Lys Lys Thr Leu ProThr Thr Phe Glu Phe Thr 65 70 75 80 Asp Ile Ala Gly Ile Val Lys Gly AlaSer Lys Gly Glu Gly Leu Gly 85 90 95 Asn Lys Phe Leu Ser His Ile Arg GluVal Asp Ala Ile Cys Gln Val 100 105 110 Val Arg Ala Phe Asp Asp Asp AsnVal Thr His Val Ala Gly Arg Val 115 120 125 Asp Pro Ile Asp Asp Ile GluVal Ile Asn Met Glu Leu Val Leu Ala 130 135 140 Asp Leu Glu Ser Val GluLys Arg Leu Pro Arg Ile Glu Lys Leu Ala 145 150 155 160 Arg Gln Lys AspLys Thr Ala Glu Met Glu Val Arg Ile Leu Thr Thr 165 170 175 Ile Lys GluAla Leu Glu Asn Gly Lys Pro Ala Arg Ser Ile Asp Phe 180 185 190 Asn GluGlu Asp Gln Lys Trp Val Asn Gln Ala Gln Leu Leu Thr Ser 195 200 205 LysLys Met Leu Tyr Ile Ala Asn Val Gly Glu Asp Glu Ile Gly Asp 210 215 220Asp Asp Asn Asp Lys Val Lys Ala Ile Arg Glu Tyr Ala Ala Gln Glu 225 230235 240 Asp Ser Glu Val Ile Val Ile Ser Ala Lys Ile Glu Glu Glu Ile Ala245 250 255 Thr Leu Asp Asp Glu Asp Lys Glu Met Phe Leu Glu Asp Leu GlyIle 260 265 270 Glu Glu Pro Gly Leu Asp Arg Leu Ile Arg Thr Thr Tyr GluLeu Leu 275 280 285 Gly Leu Ser Thr Tyr Phe Thr Ala Gly Val Gln Glu ValArg Ala Trp 290 295 300 Thr Phe Lys Gln Gly Met Thr Ala Pro Gln Cys AlaGly Ile Ile His 305 310 315 320 Thr Asp Phe Glu Arg Gly Phe Ile Arg AlaGlu Val Thr Ser Tyr Asp 325 330 335 Asp Tyr Val Gln Tyr Gly Gly Glu SerGly Ala Lys Glu Ala Gly Arg 340 345 350 Gln Arg Leu Glu Gly Lys Glu TyrIle Met Gln Asp Gly Asp Ile Val 355 360 365 His Phe Arg Phe Asn Val 37021 base pairs nucleic acid single linear not provided 5 ATGTAGATATTTATATTGCT G 21 21 base pairs nucleic acid single linear not provided 6CTAATAACAA TCACTTCAGA G 21 25 base pairs nucleic acid single linear notprovided 7 GCTCCTAAAA GGTTACTCCA CCGGC 25

What is claimed is:
 1. An isolated polynucleotide segment comprising: afirst polynucleotide sequence, wherein the first polynucleotide sequenceis selected from the group consisting of: (a) a polynucleotideconsisting of SEQ ID NO:1; and, (b) a nucleic acid sequence identical tothe polynucleotide of (a) except that, over the entire lengthcorresponding to the polynucleotide of (a), up to thirty nucleotides aresubstituted, deleted or inserted for every 100 nucleotides of thepolynucleotide of (a); and, wherein the first polynucleotide sequence isnot chromosomal DNA and wherein the nucleic acid sequence detectsStaphylococcus aureus by hybridization.
 2. The isolated polynucleotidesegment of claim 1, wherein the first polynucleotide sequence comprisesa nucleic acid sequence identical to the polynucleotide of (a) exceptthat, over the entire length corresponding to the polynucleotide of (a),up to ten nucleotides are substituted, deleted or inserted for every 100nucleotides of the polynuclcotide of (a).
 3. The isolated polynucleotidesegment of claim 1, wherein the first polynucleotide sequence comprisesa nucleic acid sequence identical to the polynucleotide of (a) exceptthat, over the entire length corresponding to the polynucleotide of (a),up to five nucleotides are substituted, deleted or inserted for every100 nucleotides of the polynucleotide of (a).
 4. A vector comprising theisolated polynucleotide segment of claim
 1. 5. An isolated host cellcomprising the vector of claim
 4. 6. A vector comprising the isolatedpolynucleotide segment of claim
 2. 7. An isolated host cell comprisingthe vector of claim
 6. 8. An isolated polynucleotide segment, comprisinga first polynucleotide sequence, wherein the first polynucleotidesequence hybridizes to the full complement of SEQ ID NO:1, wherein thehybridization conditions include incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA,followed by washing in 0.1×SSC at 65° C.; wherein the firstpolynucleotide sequence is identical to SEQ ID NO:1 exccpt that, overthe entire length corresponding to SEQ ID NO:1, up to rive nucleotideare substituted, deleted or inserted for every 100 nucleotides of SEQ IDNO:1; wherein the first polynucleotide sequence is not chromosomal DNAand wherein the nucleic acid sequence detects Staphylococcus aureus byhybridization.
 9. The isolated polynucleotide segment of claim 8,wherein the first polynucleotide sequence is identical to SEQ ID NO:1except that, over the entire length corresponding to SEQ ID NO:1, up tothree nucleotide are substituted, deleted or inserted for every 100nucleotides of SEQ ID NO:1.
 10. An isolated polynucleotide segmentcomprising the full complement of the entire length of the nucleic acidsequence of claim 1, wherein the full complement of the entire length ofthe nucleic acid sequence of claim 1 is not genomic DNA and detectsStaphylococcus aureus by hybridization.
 11. An isolated polynucleotidesegment comprising the full complement of the entire length of thenucleic acid sequence of claim 2, wherein the full complement of theentire length of the nucleic acid sequence of claim 2 is not genomic DNAand detects Staphylococcus aureus by hybridization.
 12. An isolatedpolynucleotide segment comprising the full complement of the entirelength of the nucleic acid sequence of claim 3, wherein the fullcomplement of the entire length of the nucleic acid sequence of claim 3is not genomic DNA and detects Staphylococcus aureus by hybridization.13. An isolated polynucleotide segment comprising the full complement ofthe entire length of the nucleic acid sequence of claim 8, wherein thefull complement of the entire length of the nucleic acid sequence ofclaim 8 is not genomic DNA and detects Staphylococcus aureus byhybridization.
 14. An isolated polynucleotide segment comprising thefull complement of the entire length of the nucleic acid sequence ofclaim 9, wherein the full complement of the entire length of the nucleicacid sequence of claim 9 is not genomic DNA and detects Staphylococcusaureus by hybridization.